Strategies & Considerations for Bioanalytical Method Development and Validation Using LCMS / MS - A Review

About Author: Rahul Sharma (M.Pharm - Pharmaceutics)
Lord Shiva College of Pharmacy, Sirsa, Haryana (India)

Bioanalysis, employed for the quantitative determination of drugs and their metabolites in biological fluids, plays a significant role in the evaluation and interpretation of bioequivalence, pharmacokinetic (PK), and toxicokinetic studies. The quality of these studies, which are often used to support regulatory filings, is directly related to the quality of the underlying bioanalytical data (1).  It is essential to employ well-characterized and fully validated analytical methods to yield reliable results which can be satisfactorily interpreted as well as to emphasize that each analytical technique has its own characteristics, which will vary from analyte to analyte (2).

Reference ID: PHARMATUTOR-ART-1132

The development of the sound bioanalytical method is of paramount importance during the process of drug discovery and development culminating in marketing approval (4). It is a set of various procedures, mainly  used for the determination of drugs and their metabolites in biological matrices such as urine, plasma, and serum.

Method development starts with literature survey of the molecule in which we find the nature of the molecule its pKa, solubility, molecular weight etc. Accordingly we select the appropriate chromatographic technique to determine the initial conditions of the separation. Methods for analyzing drugs by HPLC and LCMS-MS can be developed, provided one has knowledge about the nature of the sample, namely, its molecular weight, polarity, ionic character, pKa values and the solubility parameter. Method development cannot be standardized across the board because method development is unique and specific for each drug candidate. It also depends on the nature of the sample, sensitivity required etc (3,5).

Steps and Considerations for  Method Development
Before beginning method development process, we need to review certain parameters and continue accordingly, inorder to take those parameters under consideration for an effective and suitable method for a particular analyte (6). The various steps and procedures  which plays a vital role in the method development process of an analyte are:

a.) Information about the sample
The knowledge about the sample can provide valuable clues for the best choice of initial conditions for a chromatographic separation. Depending on the use made of this sample information, different approaches to LCMS method development are possible. The necessary information about an analyte before beginning the method development process are (6):

· Chemical structure (functionality) of compounds
· Molecular weight
· pKa value
· Sample Solubility and stability
· Toxicity, purity
· Concentration range of compounds in samples of interest
· Metabolites

b.) Chromatographic Approaches
By a valuable collection of information about the sample, we can easily select the chromatography method for the analyte. Different approaches are:

· First Reversed phase chromatography should be tried.
· Than normal phase chromatography should be tried if reverse phase fails.
· Ion pair chromatography is tried for ion forming compounds but before going for ion pairing, ion suppression by pH control reverse phase chromatography should be tried should be tried (3,6,7).



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